|
https://www.abbs.info ISSN 0582-9879 |
|
Mini Review |
Methods
for Structural and Functional Analysis of an RNA Hexamer of Bacterial Virus
phi29 DNA Packaging Motor
GUO
Peixuan
(
Department of Pathobiology and Purdue Cancer Center, Purdue University, West
Lafayette, IN 47907, USA )
Abstract During multiplication and maturation, the
lengthy genomic DNA of dsDNA viruses is translocated with remarkable velocity
into a limited space within the procapsid and packaged to crystalline density.
A viral DNA-packaging motor accomplishes this energy consuming motion task. An
RNA molecule of bacterial virus phi29 has been found to be a vital component of
the DNA-packaging motor. Six pRNAs form a hexagonal complex to gear the DNA
translocating machine using a mechanism similar to the driving of a bolt with a
hex nut. Sequential action of six RNA molecules to drive the motor is similar
to the consecutive firing of six cylinders of a car engine. This article
reviews the structure of pRNA to demonstrate that its structure plays a vital
role in its function, and focuses on methods and unique approaches that lead to
the elucidation of pRNA structure.
Key
words phi29；viral DNA packaging；bio-motor；molecule motor；RNA 3D
structure
The amazing diversity
in RNA function is attributed to the variety of RNA species and the flexibility
in RNA structure. To elucidate how
RNA molecules perform their versatile and novel functions, it is crucial to understand the
principles and rules that regulate RNA folding. Due to its complexity and versatility, clarifying the criterion for RNA
folding and determining RNA structure is an arduous task.
One prominent feature
in the assembly of all linear ds-DNA viruses is that their lengthy genome is
packed with a swift velocity into the pre-formed protein coating and packaged
to a near crystalline density (for review, see references[1―5]).
This DNA motion, which is an
energetically unfavorable process,
is accomplished by an ATP-hydrolyzing motor involving a connector and
two nonstructural components with certain characteristics typical of ATPases.
In phi29, one of the nonstructural
proteins for DNA packaging is an RNA (pRNA) molecule[6―9].
The connector is a 12-subunit hollow truncated cone cylinder having a central
channel with a diameter of about 4-nm through which DNA enters the procapsid
during packaging[10, 11]. The 120-base pRNA (Fig.1) encoded by the
virus, binds to the connector[12,
13] and is not present in the mature phi29 virion. The requirement for
pRNA in phi29 assembly appears to be very specific in that pRNAs from other
phages cannot replace the phi29 pRNA in in vitro packaging assays[14]
and that a single base mutation can render the pRNA inactive[15].
Fig.1 pRNA secondary structure
adapted
from[56] with
permission from J Biol Chem.
Six copies of pRNA
form a hexameric complex which serves as the essential component of the DNA
translocating motor[16―21].
DNA packaging is completely blocked when one of the six slots is occupied by an
inactive pRNA with a mutation at the 5′
or 3′ end[17, 18] since pRNA is
associated with procapsids during the DNA translocation process.
Phi29 is the most
efficient and the best-characterized in vitro DNA packaging model
system. Most essential components required to reconstitute the phi29 in
vitro DNA packaging activity have been well defined. Direct force
measurements have shown that the phi29 packaging motor is the most powerful of
all reported molecular motors,
producing a force of 57 pico-Newtons[22, 23]. In
addition, the crystal structure of
the 12-subunit (gp10) connector has been solved[11, 24]. One
nonstructural component is a protein,
gp16, that has been shown
to contain consensus ATP-binding domains[25] and is able to
hydrolyze ATP. It has been found that one ATP is needed to package two base
pairs of DNA[25].
This review will focus
on the work produced in the author’s laboratory and will concentrate on methods
and approaches that were used to investigate pRNA structure and function.
1
The role of pRNA in phi29 DNA packaging
In 1997, a
comprehensive paper was published to describe the role of phi29 pRNA in DNA
packaging[16] which provided an elegant model for explaining the
mechanism of connector (portal vertex) rotation and the quantification of
energy (ATP) usage[16]. The model presented advocated that phi29 DNA
packaging is accomplished by a mechanism similar to driving a bolt with a hex
nut, which consists of six
DNA-packaging pRNAs (Fig.2).
Recent research[11,19，20,22,23]
in this area supports the conclusion about consecutive action of six pRNAs in
driving the DNA rotation machine.
Fig.2 A model to depict the sequential action
of pRNAs in phi29 DNA packaging motor
The hexagon represents the phi29
connector and the surrounding pentagon represents the capsid. Six protrusions
represent six pRNAs. The variable pRNA patterns portray the pRNA in serial
energetic states. For example,
pRNA 4 in panel A is in a contracted conformation, and pRNA 1 is in a relaxed
conformation. Arrows marks the
different transition states of pRNA 1. Steps A to G show the six steps of
rotation. Each step rotates 12°, since a five to six-fold symmetry
mismatch generates 30 equivalent positions, and 360°/30
= 12°. The portal vertex turns 72°
after six steps. For example, pRNA 1 moves from vertex a in A to
vertex b in G, and rotates 72°.
Each step consumes one ATP to induce one conformation change of pRNA, and six ATPs are used for the
transition from one vertex to another. 30 ATPs are used for each 360°
rotation (Reprinted from[16] with permission from J Virology).
All icosahedral
viruses contain a five-fold symmetrical capsid vertex. In bacteriophage
phi29, the connector is embedded
within this five-fold symmetrical area of the capsid, while six copies of pRNA bind to the connector[16, 26]
Rotation of the hexameric pRNA (and connector) within the 5-fold symmetrical
environment could constitute a mechanical motor in which rotation may be
facilitated by the symmetry mismatch. The relative motion of the two rings
could thus produce a driving force to translocate viral DNA into the procapsid.
Similar to a car engine in which the six cylinders fire sequentially, the sequential action of six pRNAs is
one way to achieve the turning of this DNA packaging motor. An engine could not
run continuously if the cylinders fired at the same time (Fig.2).
Likewise, in the case of
phi29, sequential action of the
six pRNAs sitting on the connector may drive rotation of the connector. The pRNA may collaborate with the viral
DNA-packaging enzyme gp16 to perform this rotation job. By sequential action, it is meant that the multiple pRNAs
involved in DNA packaging appear to act in a step-by-step process, with each pRNA exerting its individual
effect alternatively (Fig.2).
The pRNA contains two
functional domains： one
for connector binding and the other for DNA translocation. Mutagenesis as well as chemical and
nuclease probing have revealed that the pRNA binds to the connector leaving the
essential 5′/3′domain
free for interaction with other components, such as gp16,
DNA-gp3 or other components on the procapsid. It has been reported that
the C18C19A20 bulge of the pRNA is
solvent-exposed when pRNA is bound to procapsid[27] and is critical
for DNA translocation[16, 28―31].
The C18C19A20 bulge might be directly involved
in interacting with ATP, gp16, DNA-gp3 or capsid components. It is predicted
that pRNA is part of an ATPase and possesses at least two conformations；
a relaxed and a contracted one. Alternating between
contraction and relaxation driven by ATP hydrolysis, each member of the hexameric RNA complex helps rotate the
translocating machine.
The requirement of an
intermolecular loop/loop interaction between individual pRNA molecules during
DNA packaging has led to the belief that pRNA forms a hexamer[19, 20]
and supports a pRNA sequential action model. The pRNAs may need to communicate with each other during DNA
packaging to ensure that the motion is consecutive. Inter-pRNA interactions via loops might serve as a link to
pass a signal to adjacent pRNAs,
regulating sequential conformational changes and/or interactions. Thus
base pairing between the right and left-hand loops might be necessary to
transfer a conformational change from one pRNA to an adjacent one.
2
Establishment of systems to facilitate functional analysis of pRNA
Four major approaches
have been undertaken in this lab to study the structure and function of pRNA.
The first approach was
the development of a highly sensitive in vitro phi29 virion assembly
system for the assay of pRNA activity[32, 33]. With this
system, up to 5×109
infectious virions per ml can be obtained in the presence of pRNA, yet not a single infectious virion is
detected in the absence of pRNA.
Therefore, a system with a
dynamic range of more than 9 orders of magnitude and with a sensitivity level
of as few as 0 infectious virions can be used for the analysis of pRNA
structure and function.
The second approach in
analyzing pRNA function was the construction of circularly permuted pRNAs
(cp-pRNA) in which any internal base of the pRNA could be reassigned to serve
as new 5′–
or 3′-termini[28, 34] (Fig.3). The
circular permutation system greatly facilitated the construction of mutant pRNA
via PCR and enabled the labeling of any specific internal base by
radioisotopes, fluorescence[35]
or photoaffinity agents.
Fig.3 Tandem DNA for the synthesis of
circularly permuted pRNA (cp-pRNA)
Adapted
from[34] with permission from Virology.
The third approach
undertaken was the construction of mutant pRNAs that were inactive in DNA
packaging, but competent to
compete with wild type pRNA for procapsid binding[16―18,
29, 36, 37].
The fourth approach to
analyze pRNA structure and function was the design of new methods to determine
the stoichiometry of pRNA in DNA packaging[18, 19, 38]. To determine
the role of pRNA in DNA packaging,
it is crucial to know how many copies of the pRNA are involved in each
DNA packaging event. Three novel
methods have been developed to determine the stoichiometry of the pRNA, and have led to the conclusion that six
pRNAs are present in each DNA translocating motor. These methods include： (a), binomial distribution (Yang Hui Triangle)[18, 38]
(Fig.4)； (b), comparing slopes of concentration dependence[18, 33]； and (c), finding the common multiple of 2, 3, and 6 by
using a set of two interlocking pRNAs,
three interlocking pRNAs and six inter-locking pRNAs[19].
Fig.4 Stoichiometry determination by binomial
distribution
Z represents the total pRNA number, initially assigned a theoretical value
from 1 to 12, per procapsid
to be determined. The empirical curve from mutant pRNA P8/P4 falls between the theoretical curves for Z = 5 and Z = 6 (Reprinted from[18]
with permission from J Virology).
3
Studies on pRNA structure
Although nucleotide
derivatives have been found in RNA,
the primary sequence of the RNA molecule is nevertheless as simple as
DNA, since both are composed of
four nucleotides. All DNA molecules appear as double helices, while RNA has a diverse structure.
Intriguingly, small RNA
molecules, containing only the
four nucleotides A, G, C, and U, exhibit
versatile biological functions.
Such versatility is ascribed to the flexibility and complexity in RNA
structural folding. NMR and X-ray crystallography have been used to obtain a
physical tertiary structure of RNAs.
Currently, NMR can only be
applied to an RNA molecule with a size of less than 40 nucleotides. X-ray crystallography of structural
RNAs has proven difficult. The
difficulty, uncertainty and
time-span in obtaining a diffractable RNA crystallographic structure, as well as the impossibility of using
NMR for large RNAs, compel the use
of alternative approaches to obtain information on RNA structures.
3.1
Genetic analysis by truncation,
insertion, deletion and
mutation
The establishment of
the highly sensitive in vitro phi29 assembly system (Section 2) greatly
facilitated the genetic analysis of pRNA structure[32, 33]. Taking
advantage of the circularly permuted pRNA system (Fig.2) (Section 2)[28,
34], the technique of
two-step PCR, and the relatively
small size of the pRNA (120 bases),
mutant pRNAs can be easily constructed with truncation, deletion, insertion and mutation targeting any desired position[29].
A plasmid DNA with two tandem RNA coding sequences linked with three bases AAA
were used as templates to generate PCR DNA fragments with primer pairs
containing either the T7 or SP6 promoter and mutations to pRNA. The DNA
fragments from PCR were used to transcribe mutant or circularly permuted pRNAs in
vitro with either T7 or SP6 RNA polymerase. In combination with the
aforementioned in vitro assembly assay system, dozens of mutant pRNAs can be obtained and tested in one or
two weeks.
By the use of the truncation
and deletion techniques, it was revealed that three nucleotides, U72U73U74,
predicted to form a bulge located at a three-helix junction (Fig.2), function to provide flexibility in pRNA folding[28]. Three
other nucleotides, C18C19A20,
were shown to be present on the surface of the pRNA as a bulge that is not involved in procapsid binding but
is essential for DNA packaging[30].
3.2
Phylogenetic analysis
Phylogenetic analysis
of RNA is used to compare the sequences of RNA molecules with identical or
similar functions from different species. A common secondary structure for RNA
molecules with a similar function is deduced from such analyses. The theory
behind such logic is that RNA structure plays a critical factor in RNA
function. Nature would select the
most stable molecule with the best-fit structure or with acceptable base
co-variations. Later on, such
phylogenetic analysis of species from nature would be expanded into molecules
made artificially, such as complementary
modification or SELEX that will be described below.
Phylogenetic analysis
revealed that pRNAs from bacteriophages SF5, phi29,
PZA, M2, NF, GA1 and B103,
which have a very low sequence identity and few conserved bases, very
impressively show similar predicted secondary structures[14, 29].
The requirement for pRNA in phi29 assembly is very specific in that pRNAs from
other phages cannot replace the phi29 pRNA in in vitro packaging[14]
and that a single base mutation can render the pRNA completely inactive[15].
Thus, similar structures do not
translate into identical function. Interestingly, phylogenetic analysis revealed that the right (upper) loop
of each pRNA was complementary to the left (lower) loop within the same
molecule[29]. Complementary modification studies reveal that the
pairing is inter-molecular[19, 20] rather than intra-molecular
(pseudoknot[39]) and that two G/C pairs are sufficient to mediate
the interaction[19, 20].
3.3
Complementary modification
Ano ther approach to
confirm base-pairing in predicted RNA structure is complementary modification.
Before the conclusion that “G
pairs to C”
in an RNA structure is drawn, at
least three mutants should be constructed and analyzed. First, mutants with either the G changed to A (or U) or the C
changed to U (or A) should be inactive.
In addition, a mutant with
both the Gs changed to As (or Us) and the Cs changed to Us (or As) should restore
the activity.
Computer predictions
of the phi29 pRNA secondary structure[40] showed that the 5′
and 3′ ends are paired. An extensive series of
helix disruptions by base substitutions almost always resulted in the loss of
DNA packaging activity. Additional
compensatory mutations that restored the predicted base pairings rescued the
activity of pRNA[15, 28, 39]. Such complementary modification has
led to the conclusion that bases 1―3
are paired with bases 117―115；bases
7―9 are paired with bases 112―110；bases
14―16 are paired with bases 103―101；and
bases 76―78
are paired with bases 90―88
(Fig.1). This second site suppression confirmed the existence of a helical
structure that is essential for pRNA function.
Complementary
modification has also been used to study inter-pRNA loop/loop interactions in
dimers[19, 20, 29]. A series of mutant pRNAs carrying mutated right
and/or left-hand loop sequences were constructed such that loop sequences were
non-complementary. Each inactive mutant was mixed with another inactive mutant
such that the loop sequences were complementary in trans, allowing the
formation of intermolecular base pairing. All mutant pRNAs that had unpaired
right and left loops, such as pRNA A-b′,
were inactive in phi29 assembly when used alone. However, when two inactive pRNAs that were
trans-complementary in their right and left loops, for example pRNA A-b′and
B-a′, were mixed in an equimolar ratio, full
activity was restored. The observed activity of a mixture of two inactive
mutant pRNAs confirmed that the right loop interacted with the left loop intermolecularly
to form an RNA dimer.
3.4
Chemical modification
Chemical modification
was employed to probe pRNA structure. The modifying agents used include
dimethyl sulfate (DMS), which methylates A at N1, G at N7 and C at N3[41,
42]；kethoxal,
which modifies G at N1 and N2[43]；and
1-cyclohexyl-3-(2-morpholinoehtyl)-carbodiimide metho-p-toluene sulfonate
(CMCT), which attacks U at N3 and G at N1[41―43].
In principle, only unpaired bases are susceptible to chemical attack. The
chemicals alter unpaired specific functional groups of RNA bases and thus
provide information regarding base pairing, base stacking, and the tertiary
interactions of specific bases within an RNA. Locations of modified bases can
be identified by primer extension with reverse transcriptase[41, 44].
Chemical modification of a base is a good indication that the base is unpaired
and that the specific functional group is solvent-exposed, and thus is a
possible candidate for intermolecular interactions. Lack of modification will
most likely be due to base pairing, especially in helical regions, but may also be the result of tertiary
interactions or non-canonical base-base,
base-sugar, or base-phosphate interactions[43] in loop or
bulge regions. Chemical modification data can provide information on base
accessibility, which is helpful in
assessing predicted secondary structures,
evaluating 3-D molecular models,
and analyzing RNA/protein interactions.
Phi29 pRNAs including
various mutants have been modified with DMS, CMCT, and
kethoxal[27, 41, 43]. Chemical modification showed that the sequence
C18C19A20, which is essential for DNA packaging but dispensable for
procapsid binding, is accessible
to chemicals in monomers and dimers as well as procapsid-bound pRNA[27,
30]. These results indicate that CCA, though not involved in procapsid binding[28, 31], is present on the surface of the pRNA
as a bulge which may interact with other DNA packaging components[30]
(Fig.5). This conclusion is supported by mutation studies on the CCA bulge.
Fig.5 Direct observation of pRNA three-dimensional structure with
cryo-AFM (atomic force microscopy) (A and B) and shape comparision with
computer models (C and D). E and F are drawings to depict the structure of
monomers and dimers, respectively
Reprinted
from[27] with permission from RNA.
As noted earlier, the
right (upper) loop sequence A45A46C47C48
and left (lower) loop sequence U85U84G83G82
of pRNA monomers are accessible to chemicals, as predicted by computer folding
algorithms. However, when specific mutant pRNAs designed to form dimers were
analyzed by chemical modification, the same sequences were protected from
modification. Normally, pRNA B-a′would
be able to interact with A-b′to
form dimers. This lack of reactivity of the bases indicates that the loop
sequences are involved in base pairing, and confirms that these bases are
involved in inter-pRNA interaction[19, 20].
3.5
Chemical modification interference
Chemical modification
interference has been performed to determine which pRNA bases are involved in
dimer formation. The monomer pRNA B-a′was
treated with either DMS or CMCT and then mixed with unmodified monomer A-b′in
order to test its competency in dimer formation. If the base is involved in
dimer formation, chemical modification of this base could interfere with the
ability of pRNA B-a′to
form a dimer with pRNA A-b′,
and thus this pRNA will be present in a fast migrating band representing
monomers in native gels. Chemical modification was performed, and RNAs (both
fast and slow migrating corresponding to pRNA monomers and dimers,
respectively) were isolated from gels. After isolation, both monomers and dimers were subjected
to primer extension to identify the modified bases. The concentration of the
chemicals was titrated to ensure that on the average only one base per pRNA was
modified[45]. The general theory behind the experiment was that a
pRNA B-a′containing
an interfering modified base would appear in the fast migrating monomer
band, while pRNA B-a′containing
a non-interfering modified base would appear in the slower migrating dimer
band. Chemical modification interference analysis reveals that bases U54,
G55, U59, C65, A66, A68,
U69, A70, C71, C84, C85,
C88, A89, A90 and C92 interfered
with dimer formation, and thus are involved in dimerization, while bases 72―81
were not involved[45] as shown in the computer model of dimer
(Fig.6).
Fig.6 Computer models of pRNA monomer (A), dimer (B), hexamer (C),
and pRNA/connector complexes (E and F). D is the crytall structure of connector
11, 72
Reprinted
from[56] with permission from J Biol Chem.
3.6
Photoaffinity crosslinking by psoralen
The chemical psoralen
can intercalate into RNA or DNA helices and, upon irradiation with 320―400
nm light, freeze (in helix or
pseudoknot) uridines of RNA or the thymidines of DNA by covalent attachment46
if they are in close proximity (in helix or pseudoknot)[47, 48]. The
sites of crosslinks can be determined by primer extension[49] and/or
mung bean nuclease treatment[50]. The psoralen derivative, AMT (4′-aminomethyl-4,
5′, 8-trimethyl psoralen), was used to
crosslink pRNA due to its solubility[49]. Psoralen crosslinks only
RNA or DNA but not protein, which
is different from the azido group (see below) which crosslinks non-specifically
to both protein and nucleic acids. Psoralen, however, can
induce intra-molecular crosslinks within the pRNA even in the presence of other
proteins, such as procapsid or gp16. Thus, pRNA conformations in different environments
can be detected. Psoralen
crosslinks can also be reversed by 254 nm irradiation. With the use of a
2-dimensional gel electrophoresis[46, 48, 51] and 5′-end
radiolabeled cp-pRNAs, pRNA
conformational change in the presence of different packaging components can be
investigated. Psoralen
crosslinking experiments revealed that pRNA had at least two conformations――one
that was able to bind procapsid and the other that was not able to bind. In the absence of Mg2+, the region comprising bases C67
to A70 was in close proximity to bases U31 to U36, since these two areas were crosslinked
together by psoralen[49].
3.7
Photoaffinity crosslinking with GMPS/Aryl azide
Aryl azides contain
functional groups that are chemically inert in the absence of light, but can be
converted to a reactive nitrene after long wavelength UV irradiation[52,
53]. Thus, aryl azides can be incorporated into RNA to obtain structural
data[54]. Aryl azide has been specifically attached to the 5′-end
of pRNAs or cp-pRNAs. For this 5′-end
labeling, 5′-thiophosphate
pRNA or cp-pRNA is synthesized by in vitro transcription in the presence
of excess GMPS (guanine-monophosphorothioate) over GTP[53]. GMPS is
an efficient primer in RNA synthesis with T7 RNA polymerase but cannot be used
by this enzyme for chain elongation. The 5′-thio-pRNA
and the 5′-thio-cpRNAs
are then treated with azidophenacyl bromide to produce the 5′-azido-pRNA
and 5′-azido-cp-pRNAs, respectively, by the nucleophilic displacement of bromine[53].
The azido group is converted to a reactive nitrene by long wavelength UV
irradiation, which is then
inserted into nearby bonds resulting in covalent crosslinks[52].
Since it is possible to generate active cp-pRNAs by assigning certain internal
sites of the pRNA as new 5′–
and 3′-termini (Section 2)[28, 34], specific
internal bases of the pRNA have been uniquely labeled with photoaffinity
crosslinking agents to analyze inter- and intra-molecular interactions. When necessary, the 5′-end
of the RNA can also be labeled with [32P]. Crosslinked RNAs were
separated from uncrosslinked RNAs by denaturing gel electrophoresis, and crosslink sites were determined by
primer extension[45, 55]. Bases identified as crosslink sites by
primer extension indicate that these bases are in close proximity to the
photoagent labeled base. The use of cp-pRNAs allows the identification of
intra-molecular contacts throughout the pRNA molecule, and such data have been used as
distance constraints in molecular modeling studies[28, 34, 45, 55]
(Section 3.13).
Intra-molecular
crosslinking of monomers[45] revealed that G108 neighbors
C10 and G11；G75
is near bases 26―30, while G78 is near U31.
The azidophenacyl group is only 0.9 nm in length, but experimental data has demonstrated that the
cross-linking group can reach distances of 1.2 nm (Norman Pace,
personal communications). These distances have been used as constraints
in the computer modeling of the pRNA monomer structure (Fig.7).
Fig.7 Comparison of chemical modification
patterns of monomer (A) and dimer (B)
The black arrow, gray square, and
double-lined arrow indicate a strong, moderate, and weak modification of
bases, respectively. C is a model
to portray the formation of dimer. The four base-pairs (45―48/85―82
in gray boxes) were modified in monomers, but were protected from chemical
modification in dimers (Adapted from[27, 45, 56] with permission
from RNA and J Biol Chem).
Intermolecular
crosslinking of dimers[55] was also achieved. Several studies
revealed that G82 is in close proximity to G39, G40, A41, C49,
G62, C63, and C64[55]. Data from these
crosslinking experiments have been used as distance constraints in molecular
modeling of pRNA dimers[56] (Fig.7).
3.8
Photo-crosslinking by phenphi
Unlike psoralen, phenphi[(cis-Rh(phen)(phi)Cl2+
(phen = 1, 10-phenanthroline；and
phi = 9, 10-phenanthrenequinone diimine)] induces covalent bonds between guanosine
bases upon UV activation. Phenphi has also been shown to crosslink pRNA and has
revealed the close proximity of bases G75, G28 and G30
of pRNA[57].
3.9
Ribonuclease probing
Some ribonucleases are
sensitive to RNA secondary structure. For example, RNases T1 (specific for GpN
linkages), U2 (specific for ApN
linkages), and S1 prefer to cleave single-stranded RNA. Nuclease V1 is specific
for double stranded RNA. End-labeled pRNA and cp-pRNA in various solutions
containing Mg2+ or procapsid individually or in combination, have been probed by T1, U2 or V1 nucleases[14, 49].
T1 and V1 were used to
distinguish the loops and helices of four RNAs with similar function[14].
RNase footprinting has also been performed to detect the sequences that bind
procapsid[49, 58]. In addition, T1 nuclease has been used to study
changes in pRNA conformation[49]. Since the activity of RNase T1 is
Mg2+ independent, this enzyme was used to investigate the
conformational change of pRNA in the presence or absence of Mg2+[49].
A Mg2+-induced
pRNA conformational change was verified by T1 ribonuclease probing[49].
The pattern of partial digestion of pRNA by T1 provided strong evidence for the
presence of two conformations, dependent on either the presence or absence of
Mg2+. Without Mg2+, strong cleavages by T1 were seen at
bases G28, G30, and G34. While in the presence
of Mg2+, these three bases became more resistant to T1 attack, indicating
a conformational change or refolding of pRNA stimulated by Mg2+[49].
3.10 Footprinting
Foot printing is a
technique derived from nuclease probing or chemical modification and is
particularly useful in probing the interaction of RNA with proteins. The
procapsid/pRNA complex was probed with nucleases A, T1 and V1[58].
The optimal concentration of enzymes was determined empirically to ensure, on
the average, one cleavage site per RNA molecule. Results of footprinting
studies revealed that bases 22―84
were protected from enzyme digestion[58] indicating that the region
from bases 22―84
contacts the procapsid.
3.11 SELEX (systematic evolution of ligands by exponential enrichment)
In vitro
evolution is a powerful tool to study consensus elements of RNA structure and
function. Starting with a library containing pRNA sequences with random mutations
within a defined region, in vitro evolution techniques allow the selection
of pRNA variants that can bind a specific ligand. Such selection for
interacting species is based on different primary structures that can adopt the
same structural feature as wild type RNA. SELEX allows screening for
co-variation of several nucleotides and can be used to reveal noncanonical
interactions that are difficult to prove by classic genetic and biochemical
approaches[59, 60]. SELEX has been used for the selection of pRNA
sequences that bind procapsids and are involved in intermolecular loop/loop
interactions[61]. It was concluded that the wild type pRNA sequence
is the most suitable sequence for procapsid binding.
3.12 Images revealed by cryo-AFM (atomic force microscopy)
Atomic force microscopy has been used by several
investigators to detect images of RNA (Fig.8) in a denatured conformation. As
the first attempt to test whether this technique can be used to detect the 3D
structure of RNA in native conformation, cryo-AFM has been performed on the
phi29 pRNA monomer, dimer and trimer[27, 37, 45].
Fig.8 Computer model of pRNA dimer is in
accord with the results of chemical modification interference
Bases that are demonstrated to interfere
with dimer formation are shown as gray spacefill bases in the pRNA subunits.
The dimer model is in agreement with the empirical data by showing that these
bases are located at the interface of the two pRNAs of the dimer (adapted from[56]
with permission from J Biol Chem).
Cryo-AFM imaging
revealed that the pRNA monomer folds into a “check
mark-shaped” structure.
The native dimers appeared as elongated shapes (Fig.9)[27]. From
this image, it can be suggested that head to head contact is involved in dimer
formation, resulting in a complex
almost twice as long as the monomer. The trimer displays a trianglular shape
under cryo-AFM[37]. The color or illumination indicates the
thickness or height of the image but does not reflect the atom density observed
end on. The brighter or whiter color indicates the thicker or taller the image；the
darker the color, the thinner the image. The color and contrast of the image
clearly indicate that the area around the head loop (the elbow of the “check
mark”) is the thickest or tallest, which
agrees with the computer model of pRNA dimmers (see below). Cryo-AFM images of
the fused dimer, which is a pRNA
construction consisting of two tandemly linked pRNAs, exhibit a similar shape to the non-covalently linked pRNA
dimer[45]. The dimensions of the covalently linked fused dimer are
comparable to that of native dimer[27, 37].
Fig.9 Computer model of pRNA dimer are in
accord with the results of intermolecular azidophenacyl photoaffinity
crosslinking
G82 (in black spacefill) in
one pRNA unit is in close proximity to G39, G40, A41,
C49, G62, C63, and C64 (in gray
wireframe) of the other pRNA unit, as determined by base-specific photo
affinity crosslinking (adapted from[56] with permission from J
Biol Chem).
3.13 Computer modeling of pRNA three-dimensional structure
The goal of modeling
pRNA structure is to organize collections of structural data from
crosslinking, chemical or
ribonuclease probing, chemical
modification interference,
cryo-AFM and other genetic data into a three-dimensional form. Since a large number of structural
constraints are available, computer programs can successfully construct
three-dimensional structures[56, 62, 63].
pRNA monomer, dimer
and hexamer (Fig.8) were produced on Silicon Graphics Octane and IndigoⅡ
computers running IRIX 6.2 or 6.5,
using the programs NAHELIX,
MANIP, PRENUC, NUCLIN, and
NUCMULT[64, 65]. The modeling was performed based on the following
assumptions：(1)
All helices were modeled as regular A-form double helices. (2) Internal loops and mismatched bases
were constructed by maintaining the integrity of the double helix while
optimizing base pairing and stacking inside the loop, as suggested by most
structural data from X-ray and NMR analysis. (3) A general rule for the
modeling of the RNA hairpin loop has been proposed[66], which
involves maximal stacking on the 3′side
of the stem and enough nucleotides stacked on the 5′side
to allow loop closure, as found in the anticodon loop of tRNA. (4) Bulges less
than four bases in size were modeled either radiating out from stems to avoid
helical distortion, while larger loops were constructed protruding from the
stems or within the helical domain, causing the helical axis to bend. Parameters
for stacking energy are considered in order to decide whether bulges should be
protruding from or within the helical stems[67]. (5) Helix
untwisting or twisting, helix-helix interactions, triple base interactions[68], pseudoknots, or other higher order
structures have been built into the model at constant geometrical distances
while allowing certain torsion angle variation. The program regarding RNA
flexibility has been applied to the construction of the pRNA UUU bulge at the
three-helix junction. This three-base bulge has been found to provide
flexibility for the appropriate folding of pRNA. Conventional computer
algorithms involving the minimization of empirical energy functions have been
considered. Twelve angstroms has been considered as a maximum distance
constraint when bases are crosslinked by GMPS/aryl azide. Modified distance
geometry and molecular mechanics algorithms using simplified “pseudo
atom” representations have been considered to
generate structures consistent with data from crosslinking, chemical
modification and chemical modification interference. A constraint satisfaction
algorithm is combined with discrete representations of nucleotide conformations
to refine the disturbed area in order to ensure the normal representation of
all atoms.
X-ray crystallography
studies have revealed that the phi29 connector contains three sections, a
narrow end, a central section, and a wider end, with diameters of 6.6 nm, 9.4
nm, 13.8 nm, res-pectively (Fig.8)[11, 69]. The hexameric pRNA model
by Hoeprich and Guo[56] contains a central channel with a diameter
of 7.6 nm, that perhaps can sheath onto the narrow end of the connector to
perhaps be anchored by the central section of the connector, which is wider
than the central channel of the pRNA hexamer (Fig.8).
As noted earlier, pRNA
contains two functional domains (Fig.1)；one
for connector binding and one for DNA translocation. The connector binding
domain is located in the middle of the pRNA primary sequence, i.e. bases 23―97, and the DNA translocation domain is
located at the 5′/3′
paired ends. It has been predicted that the connector protein (gp10) contains a
conserved RNA recognition motif (RRM), located between residues 148―214
of each gp10 monomer. This region
of gp10 is located at the narrow end of the dodecameric connector that
protrudes from the procapsid[70, 71]. The hexamer model by Hoeprich
and Guo[56] complies with the aforementioned data by showing that
pRNA bases 23―97
(colored green in Fig.6C, E &
F), within the connector binding
domain, interact with the
predicted RRM motifs of the connector (Fig.2E and F in blue), while the 5′/3′
paired region (Fig.2E in red and cyan),
comprising the DNA translocation domain, extends away from the connector.
Acknowledgements I would like to thank Jane
Kovach, Dan Shu and Stephen Hoeprich for the manuscript preparation, Drs. Mark Trottier and Chaoping Chen
for critical review, Dr. Zhifeng Shao for providing his AFM images, and Dr.
Michael Rossmann for his permission to use his published connector structure.
References
1 Earnshaw WC, Casjens SR. DNA packaging by the double-stranded DNA
bacteriophages. Cell,
1980, 21：
319―331
2 Black LW. DNA packaging in dsDNA
bacteriophages. Ann Rev Microbio,
1989, 43：
267―292
3 Guo P. Introduction：
Principles,
perspectives, and potential
applications in viral assembly. Seminars in Virology (Editor’s Introduction), 1994, 5(1)： 1―3
4 Guo P. The mechanism of DNA packaging
in double stranded DNA bacteriophages. Virologica Sinica, 1988, 2： 229―246
5 Gong ZX. Molecular assembly of virus.
Acta Biochim Biophys Sin, 1998,
30(5)： 415―418
6 Guo P, Erickson S, Anderson
D. A small viral RNA is required for in vitro packaging of bacteriophage
f29
DNA. Science 1987； 236： 690―694
7 Guo P, Trottier M. Biological and biochemical properties of the
small viral RNA (pRNA) essential for the packaging of the double-stranded DNA
of phage f29.
Seminars in Virology,
1994, 5：
27―37
8 Guo P. Structure and function of
hexameric RNA to drive viral DNA packaging motor. Prog in Nucl Acid Res
& Mole Biol, 2002；
72： 415―418
9 Guo P. Inhibition of novobiocin, coumermycin A, oxolinic acid, nalidixic, ethidium bromide and ATP analogue on the packaging of f29 DNA in vitro. Virologica
Sinica, 1988, 2： 198―205
10 Jimenez J, Santisteban A, Carazo
JM, Carrascosa JL. Computer graphic
display method for visualizing three-dimensional biological structures. Science,
1986, 232： 1113―1115
11 Simpson AA, Tao Y, Leiman
PG, Badasso MO, He Y, Jardine PJ,
Olson NH et al. Structure of the bacteriophage phi29 DNA packaging
motor. Nature, 2000, 408(6813)：
745―750
12 Guo P, Bailey S,
Bodley JW, Anderson D.
Characterization of the small RNA of the bacteriophage f29 DNA packaging machine. Nucleic
Acids Res, 1987, 15： 7081―7090
13 Garver K, Guo P. Boundary of pRNA functional domains and minimum pRNA
sequence requirement for specific connector binding and DNA packaging of phage
phi29. RNA, 1997, 3： 1068―1079
14 Bailey S, Wichitwechkarn J,
Johnson D, Reilly B, Anderson D, Bodley JW. Phylogenetic analysis and secondary structure of
the Bacillus subtilis bacteriophage RNA required for DNA packaging. J
Biol Chem, 1990, 265：
22365―22370.
15 Zhang CL, Tellinghuisen T,
Guo P. Conformation of the helical structure of the 5′/3′
termini of the essential DNA packaging pRNA of phage f29. RNA, 1995, 1： 1041―1050
16 Chen C, Guo P. Sequential action of six virus-encoded DNA-packaging
RNAs during phage phi29 genomic DNA translocation. J Virol, 1997, 71(5)： 3864―3871
17 Trottier M, Zhang CL, Guo
P. Complete inhibition of virion assembly in vivo with mutant pRNA
essential for phage f29 DNA packaging. J Virol, 1996, 70： 55―61
18 Trottier M, Guo P. Approaches to determine stoichiometry of viral
assembly components. J Virol,
1997, 71：
487―494
19 Guo P, Zhang C, Chen
C, Trottier M, Garver K. Inter-RNA interaction of phage
phi29 pRNA to form a hexameric complex for viral DNA transportation. Mol
Cell, 1998, 2： 149―155
20 Zhang F, Lemieux S, Wu
X, StArnaud D, McMurray CT, Major F,
Anderson D. Function of hexameric RNA in packaging of bacteriophage
phi29 DNA in vitro. Mol Cell,
1998, 2：
141―147
21 Hendrix RW. Bacteriophage DNA packaging：
RNA gears in a DNA transport machine (Minireview). Cell, 1998, 94： 147―150
22 Smith DE, Tans SJ, Smith
SB, Grimes S, Anderson DL, Bustamante C. The bacteriophage phi29 portal motor can
package DNA against a large internal force. Nature, 2001, 413： 748―752
23 Davenport R.J. Crossover research yield
scents and sensitivity-watching a virus get stuffed. Science, 2001, 291： 2071―2072
24 Valpuesta JM, Fernandez JJ,
Carazo JM, Carrascosa JL.
The three-dimensional structure of a DNA translocating machine at 10 A
resolution. Structure Fold Des,
1999, 7：
289―296
25 Guo P, Peterson C,
Anderson D. Prohead and DNA-gp3-dependent ATPase activity of the DNA
packaging protein gp16 of bacteriophage f29. J Mol Biol, 1987, 197： 229―236
26 Hendrix RW. Summetry mismatch and DNA
packaging in large bacteriophages. Proc Natl Acad Sci USA, 1978, 75： 4779―4783
27 Trottier M, Mat-Arip Y,
Zhang C, Chen C, Sheng S, Shao Z, Guo P.
Probing the structure of monomers and dimers of the bacterial virus phi29
hexamer RNA complex by chemical modification. RNA, 2000, 6： 1-10： 1257―1266
28 Zhang CL, Tellinghuisen T,
Guo P. Use of circular permutation to assess six bulges and four loops
of DNA-packaging pRNA of bacteriophage f29. RNA, 1997, 3： 315―322
29 Chen C, Zhang C, Guo P.
Sequence requirement for hand-in-hand interaction in formation of pRNA dimers
and hexamers to gear phi29 DNA
translocation motor. RNA,
1999, 5：
805―818
30 Zhang C, Trottier M,
Chen C, Guo P. Chemical
modification patterns of active and inactive as well as procapsid-bound and
unbound DNA-packaging RNA of bacterial virus Phi29. Virology, 2001, 281： 281―293
31 Reid RJD, Bodley JW,
Anderson D. Identification of bacteriophage f29 prohead RNA (pRNA) domains
necessary for in vitro
DNA-gp3 packaging. J Biol Chem, 1994, 269：9084―9089
32 Lee CS, Guo P. A highly sensitive
system for the in vitro assembly of bacteriophage f29 of Bacillus subtilis.
Virology, 1994, 202：1039―1042
33 Lee CS, Guo P. In vitro assembly
of infectious virions of ds-DNA phage f29 from cloned gene products and
synthetic nucleic acids. J Virol 1995；69：5018―5023.
34 Zhang CL, Trottier M, Guo PX. Circularly permuted viral pRNA active
and specific in the packaging of bacteriophage f29 DNA. Virology, 1995,
207：442―451
35 Wu T, Ruan K, Liu W. A
fluorescence-labeling method for sequencing small RNA on polyacrylamide gel. Nucleic
Acids Res, 1996, 24： 3472―3473
36 Trottier M, Garver K, Zhang
C, Guo P. DNA-packaging pRNA as
target for complete inhibition of viral assembly in vitro and in vivo.
Nucleic Acids Symposium Series, 1997, 36：187―189
37 Chen C, Sheng S, Shao
Z, Guo P. A dimer as a building
block in assembling RNA. A hexamer that gears bacterial virus phi29
DNA-translocating machinery. J Biol Chem, 2000, 275(23)：17510―17516
38 Chen C, Trottier M, Guo
P. New approaches to stoichiometry determination and mechanism investigation on
RNA involved in intermediate reactions. Nucleic Acids Symposium Series, 1997, 36： 190―193
39 Reid RJD, Zhang F, Benson
S, Anderson D. Probing the
structure of bacteriophage f29 prohead RNA with specific mutations. J
Biol Chem, 1994, 269：
18656―18661
40 Zuker M. On finding all suboptimal
foldings of an RNA molecule. Science, 1989, 244：
48―52
41 Moazed U, Stern S, Noller
HF. Rapid chemical probing of conformation in 16S ribosomal RNA and 30 S
ribosomal subunits using primer extension. J Mol Biol, 1986, 187： 399―416
42 Yap LP, Musier-Forsyth K. Transfer RNA aminoacylation：
Identification of a critical ribose 2′-hydroxyl-base
interaction. RNA 1995, 1：
418―424
43 Ehresmann C, Baudin F,
Mougel M, Romby P, Ebel JP, Ehresmann B. Probing the structure of RNAs in solution. Nucleic
Acids Res, 1987, 15： 9109―9128
44 Stern S, Moazed D,
Noller HF. Structural analysis of RNA using chemical and enzymatic
probing monitored by primer extension. Meth Enzymol, 1988, 164： 481―489
45 Mat-Arip Y, Garver K, Chen
C, Sheng S, Shao Z, Guo P. Three-dimensional interaction of phi29 pRNA dimer
probed by chemical modification interference, cryo-AFM, and cross-linking.
J Biol Chem, 2001, 276：
32575―32584
46 Wassarman DA. Psoralen crosslinking of
small RNAs in vitro. Molecular Biology Reports, 1993, 17： 143―151
47 Tyc K, Steitz JA. A new interaction between the mouse 5′ external transcribed spacer of pre-rRNA
and U3 snRNA detected by psoralen crosslinking. Nucleic Acids Res, 1992, 20： 5375―5382
48 Cimino GD, Gamper HB,
Isaacs ST, Hearst JE.
Psoralens as photoactive probes of nucleic acid structure and function：
Organic chemistry,
photochemistry, and
biochemistry. Ann Rev Biochem,
1985, 54：
1151―1193
49 Chen C, Guo P. Magnesium-induced conformational change of packaging
RNA for procapsid recognition and binding during phage phi29 DNA encapsidation.
J Virol, 1997, 71： 495―500
50 Hui CF, Cantor CR. Mapping the location of psoralen crosslinks on
RNA by mung bean nuclease sensitivity of RNA-DNA hybrids. Proc Natl Acad Sci
USA, 1985, 82： 1381―1385
51 Wassarman DA, Steitz JA. Interactions of small nuclear RNA’s with
precursor messenger RNA during in vitro splicing. Science
1992, 257：
1918―1925
52 Hixson SH, Hixson SS. p-Azidophenacyl bromide, a versatile photolabile bifunctional
reagent. Reaction with glyceraldehyde-3-phosphate dehydrogenase. Biochemistry, 1975, 14： 4251―4254
53 Burgin AB, Pace NR. Mapping the active site of ribonuclease P RNA using
a substrate containing a photoaffinity agent. EMBO J, 1990, 9： 4111―4118
54 Nolan JM, Burke DH, Pace
NR. Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease
P RNA structure. Science,
1993, 261：
762―765
55 Garver K, Guo P. Mapping the inter-RNA interaction of phage phi29 by
site-specific photoaffinity crosslinking. J Biol Chem, 2000, 275(4)： 2817―2824
56 Hoeprich S, Guo P. Computer modeling of 3-D structure of pRNA
monomer, dimer and hexamer of
phi29 DNA packaging motor. J Biol Chem, 2002, 277: 20794―20803
57 Mohammad T, Chen C, Guo
P, Morrison H. Photoinduced
cross-linking of RNA by cis-Rh(phen)2Cl2+ and cis–
Rh(phen)(phi)Cl2+：a
new family of light activatable nucleic acid cross-linking agents. Bioorg
Med Chem Lett, 1999, 9： 1703―1708
58 Reid RJD, Bodley JW,
Anderson D. Characterization of the prohead-pRNA interaction of
bacteriophage f29.
J Biol Chem, 1994, 269：
5157―5162
59 Ellington AD, Szostak JW. In vitro
selection of RNA molecules that bind specific ligands. Nature, 1990, 346： 818―822
60 Tuerk G, Gold L. Systematic evolution
of ligands by exponential enrichment： RNA ligands to bacteriophage T4 DNA
ploymerase. Science,
1990, 249：
505―510
61 Zhang F, Anderson D. In vitro selection of bacteriophage phi29
prohead RNA aptamers for prohead binding. J Biol Chem, 1998, 273： 2947―2953
62 Babcock MS, Pednault EPD,
Olson WK. Nucleic acid structure analysis. Mathematics for local
cartesian and helical structure parameters that are truly comparable between
structures. J Mol Biol,
1994, 237：
125―156
63 Gautheret D, Cedergren R. Modeling the three-dimensional structure of
RNA. FASEB J,
1993, 7：
97―105
64 Massire C, Westhof E. MANIP： An interactive tool for modelling RNA. J
Mol Graph Model, 1998, 16： 197―145
65 Westhof E, Dumas P, Moras
D. Crystallographic refinement of yeast aspartic acid transfer RNA. J Mol
Biol, 1985, 184： 119
66 Haasnoot CAG, Hilbers CW, van
der Marel GA, van Broom JH, Singh UC, Pattabiraman N,
Kollman PA. On loop folding in nucleic acid hairpin-type structures. J
Biomol Struct & Dyn,
1986, 3：
843―857
67 Turner DH, Sugimoto N,
Freier SM. RNA structure prediction. Annu Rev Biophys Chem, 1988, 17： 167―192
68 Michel F, Ellington AD,
Couture S, Szostak JW.
Phylogenetic and genetic evidence for base-triples in the catalytic domain of
group I introns. Nature,
1990, 34：
578―580
69 Simpson AA, Leiman PG, Tao
Y, He Y, Badasso MO,
Jardine PJ, Anderson DL et
al. Structure determination of the head-tail connector of bacteriophage phi29. Acta
Crystallogr D Biol Crystallogr,
2001, 57(Pt 9)：
1260―1269
70 Grimes S, Anderson D. RNA Dependence of the bateriophage phi29 DNA
packaging ATPase. Mol Biol,
1990, 215：
559―566
71 Carazo JM, Donate LE,
Herranz L, Secilla JP, Carrascosa JL. Three-dimensional
reconstruction of the connector of bacteriophage f29 at 1.8 nm resolution. J
Mol Biol, 1986, 192：
853―867
Received：April
25, 2002 Accepted：May
17, 2002
The work in the
author’s laboratory is supported by NIH grants GM59944, GM60529, GM48159 and
GM46490, as well as NSF grant MCB9723923
Correspondence：Tel, (765) 494-7561；Fax, (765) 496-1795；e-mail, [email protected] Review